Yarrowia lipolytica and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to yarrowia lipolytica and application thereof. The strain of yarrowia lipolytica capable of tolerating high ammonia nitrogen concentration is obtained for the first time and can directly utilize a nitrogen source in the biogas slurry stock solution to produce the microbial protein under the aerobic and additional carbon source adding conditions, water does not need to be additionally added for dilution, the process steps of biogas slurry biological nitrogen removal and nitrogen assimilation can be effectively simplified, and the production cost is reduced. The harmless and resource utilization cost of the biogas slurry is reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a Yarrowia lipolytica and application thereof. BACKGROUND

[0002] There are two important problems in the development of large-scale and intensive breeding industry: (1) a large amount of residual biogas slurry after anaerobic fermentation of livestock manure is difficult to be effectively treated; and (2) feed protein raw materials (soybeans) required for livestock breeding are severely dependent on imports. The biogas slurry contains rich nitrogen, phosphorus, potassium and other macronutrients and other micronutrients, and also contains various amino acids, vitamins and other substances, and is a good microbial culture medium. Microbial protein is a new type of protein source, which has the advantages of high production efficiency, wide substrate source, high protein content (35% to 60%), and production not affected by regional environment and seasonal changes. Therefore, the biogas slurry can be used to produce microbial protein, and the harmless treatment of the biogas slurry and the production of microbial protein can be simultaneously realized.

[0003] However, the original biogas slurry has complex components, high ammonia nitrogen content (for example, the ammonia nitrogen concentration of chicken manure is often higher than 5000 mg / L), and high content of inhibiting substances such as organic acids and furfural, which leads to the fact that most protein-producing microorganisms cannot survive in the original biogas slurry. Therefore, the biogas slurry needs to be diluted and pretreated, and then the microorganisms can normally function. For example, the previous patents “a strain of honey yeast and a method for producing single-cell protein by treating high-ammonia-nitrogen biogas slurry” and “a strain of white geotrichum and a method for producing single-cell protein by treating high-ammonia-nitrogen biogas slurry” provided by the application group provide a strain (honey yeast and white geotrichum) that can survive in the biogas slurry diluted by 2 to 3 times and be used for producing single-cell protein, but these microorganisms cannot tolerate the original biogas slurry. Diluting the biogas slurry with water not only increases the process difficulty and treatment cost, but also increases the amount of waste water to be treated at the back end.

[0004] Therefore, it is of important practical significance and broad application prospect to screen and obtain a protein-producing microorganism that can tolerate high-concentration ammonia nitrogen and normally grow in the original biogas slurry. SUMMARY

[0005] The application aims to provide a Yarrowia lipolytica and application thereof.

[0006] To achieve the above application purposes, the technical solution adopted by the application is as follows: a Yarrowia lipolytica, which was preserved in the China General Microbiological Culture Collection Center on June 13, 2025, and the preservation number is CGMCC NO.34879.

[0007] Correspondingly, a microbial preparation prepared by using the Yarrowia lipolytica, or a microbial preparation containing the Yarrowia lipolytica.

[0008] Correspondingly, the application of the Yarrowia lipolytica or the bacterial preparation in degrading ammonia nitrogen, biogas slurry treatment and preparing microbial protein.

[0009] The application has the following beneficial effects: the existing disclosed Yarrowia lipolytica cannot directly survive in chicken manure biogas slurry raw solution, and can only tolerate ammonia nitrogen concentration of 2-3 g / L at most. The Yarrowia lipolytica of the application can tolerate ammonia nitrogen of 80 g / L at most for the first time, and can directly produce microbial protein by using the nitrogen source in the biogas slurry raw solution under the condition of oxygen and additional carbon source, without separate dilution with water, so that the process steps of biogas slurry biological denitrification and nitrogen assimilation can be effectively simplified, and the harmless and resource utilization cost of the biogas slurry can be reduced. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 It is a colony morphology diagram of Yarrowia lipolytica;

[0011] Figure 2 It is a scanning electron microscope diagram of the Yarrowia lipolytica strain;

[0012] Figure 3 It is a growth curve diagram of the Yarrowia lipolytica in YPD medium;

[0013] Figure 4 It is a growth curve diagram of the Yarrowia lipolytica in the original high ammonia nitrogen biogas slurry (ammonia nitrogen concentration 6000 mg / L) medium;

[0014] Figure 5 It is a phylogenetic tree diagram of the Yarrowia lipolytica. DETAILED DESCRIPTION

[0015] The application provides a new Yarrowia lipolytica (Yarrowia lipolytica D4), which is preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 13, 2025, the address of the preservation center is No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO. 34879.

[0016] The Yarrowia lipolytica can tolerate ammonia nitrogen of 80 g / L at most, can directly grow in untreated (including dilution treatment) biogas slurry, and can produce microbial protein by using ammonia nitrogen in the biogas slurry.

[0017] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. If not specifically indicated, the technical means used in the embodiments are conventional means familiar to those skilled in the art, and the obtained data are all average values obtained after at least 3 repetitions, and the data obtained in each repetition are all valid data.

[0018] The biogas slurry and the culture medium used in the present application are as follows:

[0019] 1. The original biogas slurry used in the present application is chicken manure biogas slurry, and the main components and initial ammonia nitrogen concentration are shown in Table 1 (measured in 3 repetitions).

[0020] Table 1. Control table of ammonia nitrogen concentration and components of chicken manure biogas slurry original solution

[0021] Indicator Replicate 1 Replicate 2 Replicate 3 Average Ammonia nitrogen (mg / L) 6360.32 6364.12 6551.88 6425.44 Glucose (g / L) 0.00 0.02 0.02 0.01 Ethanol (g / L) 0.52 0.58 0.57 0.56 Lactic acid 0.00 0.00 0.00 0.00 Acetic acid 541.64 575.87 614.46 577.32 Propionic acid 0.00 0.00 0.00 0.00 Butyric acid 369.55 506.45 445.79 440.60 Valeric acid 2520.94 2701.62 2701.43 2641.33 Caproic acid 1170.78 1304.17 1415.60 1296.85

[0022] 2. Biogas slurry culture medium (1L): after sterilization of the ultrafiltrated chicken manure biogas slurry (ammonia nitrogen concentration > 5000 mg / L) at 115℃ for 20 min, the ammonia nitrogen concentration is diluted to 3000 mg / L with sterile water, 22.5 g of glucose (C / N = 3) is added, 4M H2SO4 is used to adjust pH = 6.5, and sterile water is used to make up to 1L.

[0023] 3. YPD culture medium (1L): 10 g of yeast powder, 20 g of peptone, 20 g of glucose, and sterile water to make up to 1L.

[0024] 4. High ammonia nitrogen biogas slurry culture medium (1L): after sterilization of the ultrafiltrated chicken manure biogas slurry (ammonia nitrogen concentration > 5000 mg / L) at 115℃ for 20 min, a certain amount of ammonium sulfate is added according to the ammonia nitrogen concentration after sterilization to adjust the ammonia nitrogen concentration to 6 g / L, 30 g of glucose (C / N = 2) is added, and 4M H2SO4 is used to adjust pH = 6.5.

[0025] 5. Carbon source assimilation basic culture medium (1L): (NH4)2SO4 2.64 g, KH2PO4 2.38 g, K2HPO4 5.65 g, MgSO4·7H2O 1 g, CuSO4·5H2O 0.0064 g, FeSO4·7H2O 0.001 g, MnCl2·4H2O 0.0074 g, ZnSO4·7H2O 0.0015 g, pH = 6.5, and the amount of carbon source added is 0.5%.

[0026] 6. Nitrogen source assimilation base medium: 1% glucose, 0.02% KH2PO4, 0.05% CaCO3, 0.01% CaSO4.2H2O, 0.02% MgSO4.7H2O, 0.02% NaCl, pH=6.5, nitrogen source addition amount 0.5%.

[0027] Add agar 15 g / L to each medium base to obtain the corresponding solid medium.

[0028] Example 1: Screening and identification of strains

[0029] 1. Screening

[0030] Mix fruit, kitchen waste, domestic sewage, mash, pickles, pickled vegetables and soybean paste samples uniformly, first expand culture in YPD liquid medium at 28°C, 200 rpm for 24 h as the seed liquid of preliminary domestication. Inoculate the seed liquid in a 7 L fermenter containing glucose (22.5 g / L), sterilized biogas liquid medium (ammonia nitrogen concentration 3000 mg / L), and domestication system. The domestication culture conditions are: aeration amount 5 L / min, temperature 28°C, 7 days for one generation. Then use the bacteria liquid obtained from the first generation domestication as the seed liquid for the second generation domestication, and domesticate under the same conditions for the same time, for a total of three generations.

[0031] After domestication, dilute the bacteria liquid and spread on solid biogas liquid medium (ammonia nitrogen concentration 3000 mg / L), invert culture at 28°C for 48 h, pick different colonies grown on the plate, number and purify by streaking three times.

[0032] After obtaining the purified single colonies, culture in liquid YPD medium at 28°C, 200 rpm for 24 h to obtain the seed liquid of each strain. Inoculate each seed liquid into a 250 mL conical flask containing 50 mL biogas liquid medium with ammonia nitrogen concentration of 3000 mg / L at an inoculation amount of 10%, and culture at 28°C, 200 rpm for 48 h. Then measure OD 600 , select the strain with the highest OD 600 value, OD 600 =2.16, numbered: D4.

[0033] 2. Identification

[0034] (1) Basic physiological and biochemical characteristics: after streaking on YPD solid medium, strain D4 was cultured at 28°C for 48 h, forming round-edged regular colonies, the colonies were milky white, the outer surface was moist and shiny, easy to pick up, the colony morphology is shown in Figure 1 , and the scanning electron micrograph of the strain is shown in Figure 2 . The suitable growth temperature of the strain is 25-30°C; the tolerable pH is 3-8, and the suitable pH is 6.5.

[0035] (2) Carbon source assimilation experiment: The carbon sources tested included glucose, glycerol, ethanol, fructose, mannose, acetic acid, lactic acid, citric acid, soluble starch, sucrose, xylose, and lactose, with the addition amount of 0.5% (v / v) respectively. The negative control did not add any carbon source. The bacterial liquid was inoculated at 10% (v / v) into a 250 mL conical flask containing 50 mL of carbon source assimilation basal medium and cultured at 28°C and 200 rpm for 24 h. By comparing the initial and end point OD 600 , to determine whether the strain can assimilate carbon source, compared with the negative control, OD 600 An increase of more than 0.5 was recorded as positive "++", an increase of 0.2 to 0.5 was recorded as weak positive "+", and an increase of less than 0.2 was recorded as negative "-". The results are shown in Table 2.

[0036] Table 2 Results of carbon sources that can be utilized by strains

[0037] Carbon source Result Carbon source Result Glucose ++ Lactic acid + Glycerol ++ Citric acid + Ethanol ++ Soluble starch - Fructose ++ Sucrose - Mannose ++ Xylose - Acetic acid + Lactose -

[0038] (3) Nitrogen source assimilation experiment: The addition amount of the test nitrogen source (ammonium chloride, ammonium sulfate, potassium nitrate) was 0.5% (v / v), and the negative control was not added with any nitrogen source. After the bacterial solution was starved for 4 hours, it was inoculated with 10% (v / v) of the inoculum into a 250 mL conical flask containing 50 mL of nitrogen source assimilation basal medium and cultured at 28°C and 200 rpm for 24 hours. By comparing the initial and end point OD 600 To determine whether the strain can assimilate nitrogen sources, compared with the negative control, OD 600 An increase of more than 0.5 was recorded as positive "++", an increase of 0.2 to 0.5 was recorded as weak positive "+", and an increase of less than 0.2 was recorded as negative "-". The results are shown in Table 3.

[0039] Table 3 Results of nitrogen sources that can be utilized by strains

[0040]

[0041]

[0042] (4) Growth curve

[0043] The strain was inoculated into YPD liquid medium, cultured at 26°C and 200 rpm, and the OD was measured every 2 h. 600 , and draw a growth curve. The results are as follows Figure 3 shown.

[0044] The strain was inoculated into a high ammonia nitrogen biogas slurry culture medium with a concentration of 6000 mg / L ammonia nitrogen at a 10% inoculum (v / v) and cultured at 28°C and 200 rpm. The OD was measured every 12 h. 600 , and draw a growth curve. The results are as follows Figure 4shown.

[0045] (5) Molecular biological identification: The cultured bacterial solution was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing and identification. The ITS sequence was sequenced using the ITS1-ITS4 primer pair. The resulting sequence is shown in SEQ ID NO: 1. The resulting DNA sequence was input into NCBI for Blast search. The obtained nucleotide sequence was compared and analyzed with the corresponding sequences of similar strains included in NCBI using Clustalx and MEGA software, and a phylogenetic tree was constructed (e.g. Figure 5 The strain D4 was found to be most closely related to Yarrowia lipolytica culture CBS:7133 (identity = 99.71%). Based on its morphological, physiological and biochemical characteristics, the strain D4 was preliminarily identified as Yarrowia lipolytica.

[0046] Based on the results of comprehensive molecular identification and physiological and biochemical characteristics, the strain was identified as Yarrowia lipolytica, and was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC) on June 13, 2025. The deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC NO.34879.

[0047] Example 2: Demonstration of the ability of strains to produce single-cell protein using high-ammonia biogas slurry

[0048] The original biogas slurry was diluted 6 times and the ammonia nitrogen was adjusted to 1g / L. On this basis, ammonium sulfate was added to make the ammonia nitrogen concentration 2g / L to 80g / L, and 30g / L of glucose was added. The strain was activated and the OD was adjusted. 600 =2.0, inoculated at an inoculum size of 10% (v / v) into culture media with varying ammonia nitrogen concentrations, adjusted the initial pH to 6.5, and cultured in a shaking incubator at 26°C and 200 rpm for 72 h. After completion of the culture, the cell dry weight (CDW), culture medium pH, and the residual reducing sugar (glucose) content in the culture medium were measured. The results are shown in Table 4.

[0049] Table 4 Comparison of strain tolerance and utilization of high-concentration ammonia nitrogen biogas slurry

[0050] Initial ammonia nitrogen concentration (g / L) End pH Remaining reducing sugar (g / L) CDW (g / L) 1 2.92 0.07 10.29 2 2.91 0.07 10.30 3 2.90 0.06 10.19 4 3.09 0.06 10.53 5 3.20 0.06 10.33 6 3.22 0.07 10.27 7 3.27 0.07 10.31 9 3.33 0.07 10.27 11 3.41 0.08 9.73 13 3.44 0.08 10.01 15 3.53 0.08 9.22 17 3.60 0.35 8.14 19 3.64 0.89 8.17 21 3.74 1.28 8.03 23 4.10 1.08 9.73 25 4.15 0.91 9.64 27 4.16 0.68 9.41 30 4.15 1.12 9.30 35 6.13 9.80 8.63 40 6.52 18.05 6.99 50 6.35 18.13 6.67 60 6.65 18.41 5.73 70 6.69 18.25 4.51 80 6.72 18.36 2.63

[0051] The results show that the strain D4 can grow at 1-70 g / L or even higher ammonia nitrogen concentration, and the CDW is stable at 10 g / L or above when the ammonia nitrogen concentration is 1-9 g / L. When the ammonia nitrogen concentration is increased to 60 g / L, the CDW is 5.73 g / L, which is about 54.42% of the highest biomass (10.53 g / L at 4 g / L ammonia nitrogen concentration), indicating that the strain D4 can grow normally at 60 g / L ammonia nitrogen concentration. With the continuous increase of ammonia nitrogen concentration, the CDW gradually decreases, and when it is 80 g / L, the CDW is 2.63 g / L, which is 24.98% of the highest biomass, and the growth of the strain is greatly inhibited.

[0052] Example Three: Effect of culture conditions on the utilization of high-concentration ammonia nitrogen biogas slurry by the strain

[0053] 1. Effect of C / N: Use high-ammonia nitrogen biogas slurry medium with an initial ammonia nitrogen concentration of 6000 mg / L, and adjust the amount of glucose to make the C / N in the medium be 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, and 8, respectively. Activate the strain and adjust the OD 600 = 2.0, inoculate it into high-ammonia nitrogen biogas slurry medium with different C / N (initial pH = 6.5) at an inoculation amount of 10% (v / v), and cultivate it at 28°C and 200 rpm for 72 h. Then measure the ammonia nitrogen concentration, reducing sugar content, and CDW in the medium, and calculate the cell yield. The results are shown in Table 5.

[0054] Table 5 Growth of strain D4 at different C / N

[0055]

[0056] As can be seen from the data in the table, the CDW is the highest (11.95 g / L) when C / N = 7, the cell yield is the highest (35.93%) when C / N = 2, and the ammonia nitrogen utilization rate is the highest, and the reducing sugar is almost completely used up when C / N is 0.5-1.5. From the economic point of view, considering the CDW and cell yield, it is considered that the economic benefit of strain D4 in producing microbial protein by using original biogas slurry is the best when C / N = 2. In addition, although the same C / N and ammonia nitrogen concentration are used in this example and Example Two, the original biogas slurry in this example contains higher concentration of microbial inhibitors, so the CDW obtained in this example (Table 5, 9.45 g / L) is slightly lower than that in Example Two (Table 4, 10.27 g / L).

[0057] 2. Effect of pH: Using high ammonia nitrogen slurry medium with initial ammonia nitrogen concentration of 6000 mg / L, adding glucose of 30 g (C / N = 2). Using 4M H2SO4, 4M NaOH to adjust pH to 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 respectively. The strain was activated, adjusted OD 600 = 2.0, inoculated into high ammonia nitrogen slurry medium with different pH at inoculation amount of 10% (v / v), cultured at 28℃, 200 rpm for 72 h, then determined ammonia nitrogen concentration, reducing sugar content, pH and CDW in the medium. The results are shown in Table 6.

[0058] Table 6 Effect of different pH on strain 4

[0059]

[0060] From the data in the table, it can be seen that strain D4 can grow at pH 3.5-8, the highest CDW is 9.12 g / L at pH = 6.5, and the highest sugar utilization rate, so 6.5 is considered as the optimum pH for its growth in the original slurry.

[0061] The above-described embodiments are only to describe the preferred modes of the present application, and do not limit the scope of the present application, and various modifications, variations, modifications, replacements of the technical solutions of the present application made by those skilled in the art without departing from the design spirit of the present application shall fall within the protection scope determined by the claims of the present application.

Claims

1. A strain of Yarrowia lipolytica, characterized by: It was deposited in the General Microbiology Center of China Culture Collection Administration on June 13, 2025, with the deposit number: CGMCC NO.34879.

2. A bacterial preparation prepared using the Yarrowia lipolytica according to claim 1.

3. A bacterial preparation containing the Yarrowia lipolytica according to claim 1.

4. Use of the Yarrowia lipolytica according to claim 1 or the bacterial preparation according to claim 2 or 3 in degrading ammonia nitrogen.

5. Use of the Yarrowia lipolytica according to claim 1 or the bacterial preparation according to claim 2 or 3 in biogas slurry treatment.

6. Use of the Yarrowia lipolytica according to claim 1 or the bacterial preparation according to claim 2 or 3 in the preparation of microbial protein.

7. The use according to any one of claims 4 to 6, characterized in that: The application temperature is 25-30°C.

8. The use according to any one of claims 4 to 6, characterized in that: The pH of the application is 3-8.

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